雷帕霉素和3-甲基腺嘌呤对RAW264.7细胞中8种miRNAs表达的影响  

Effects of rapamycin and 3-methyladenine on the expression of eight miRNAs in RAW264.7 cells

作　　者：徐向荣[1] 马占兵[2] 蒋丹[1] 屈昱良[1] 王羡[1] 方媛[1] 郭乐[1] 徐广贤[1] 

机构地区：[1]宁夏医科大学临床医学院医学检验系,银川710004 [2]宁夏医科大学基础医学院医学遗传系与细胞生物学系,银川710004

出　　处：《免疫学杂志》2017年第9期800-806,共7页Immunological Journal

基　　金：国家自然科学基金(31472168);宁夏科技创新领军人才项目(2015年)

摘　　要：目的探究雷帕霉素(Rapamycin,Rapa)和3-甲基腺嘌呤(3-methyladenine,3-MA)处理对RAW264.7细胞中miR-181c,miR-101b和miR-1192等8种自噬相关miRNAs的表达水平,为研究miRNAs在细胞自噬中的功能提供理论基础。方法生物信息学预测分析与细胞自噬相关的miRNAs并构建miRNAs调控细胞自噬的网络图;分别用Rapa和3-MA对RAW264.7细胞做不同条件处理,通过实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)检测不同条件处理后RAW264.7细胞中miR-181b、miR-181c、miR-101b、miR-1192、miR-362-3p、miR-590-3p、miR-875-5p和miR-335-5p的相对表达量。结果生物信息学预测结果显示8种miRNAs可能分别靶向作用于细胞自噬通路中多个关键蛋白,对细胞自噬起到促进或抑制作用;与正常对照组相比,miR-181c、miR-101b、miR-1192、miR-362-3p、miR-335-5p、miR-875-5p在Rapa处理RAW264.7细胞组中表达显著下调(P<0.05),而在3-MA处理组则都显著上调(P<0.05);miR-181b在Rapa组表达显著上调,在3-MA组表达下调(P<0.05);miR-590-3p在Rapa组和3-MA组都无显著性变化(P>0.05)。结论 miR-181c、miR-101b等7种miRNAs可能在RAW264.7巨噬细胞自噬过程中发挥重要调控作用。This study was performed to determine the expression levels of miR-181c, miR-101b and miR-1192 etc. in mouse RAW264.7 cells and provide a theoretical basis for exploring the function of miRNAs in macrophage autophagy. Bioinformatics analysis was used to predict autophagy-related miRNAs and establish miRNAs-regulated network of autophagy. The relative expression of miR-181b, miR-181c, miR-101b, miR-1192,miR-362-3p, miR-590-3p, miR-875-5p and miR-335-5p were detected by qRT-PCR in RAW264.7 cell models pre-treated with different conditions of Rapamycin（Rapa） or 3-methyladenine（3-MA）. The results of bioinformatics showed that eight miRNAs could target key proteins in autophagy pathway, which may facilitate or inhibit autophagy function. Compared with the normal control, the expression of miR-181c, miR-101b, miR-1192,miR-362-3p, miR-335-5p and miR-875-5p in RAW264.7 cells was significantly down-regulated after Rapatreatment（P〈0.05）, while markedly up-regulated in the 3-MA treatment group（P〈0.05）; however, the opposite expression level variation of miR-181b was observedunder the same conditions as above（P〈0.05）. The expression of miR-590-3p had no change after anytreatments（P〉0.05）. In conclusion, our research suggest that, except for miR-590-3p, other seven miRNAs present remarkably differential expression and thus play critical regulatory roles in RAW264.7 mousemacrophage autophagy.

关 键 词：自噬 MIRNAS REAL-TIME PCR 

分 类 号：R392[医药卫生—免疫学]